1. Field of the Invention
The present invention relates to a method for improving the yield of a protein that is typically difficult to purify using gel filtration chromatography and a water soluble buffer. More specifically, the invention relates to a protein purification method whereby the addition of arginine to a developing solvent enables improvement in recovery yields of antibodies, hydrophobic proteins, or hydrophobic peptides.
2. Brief Description of the Related Art
Knowledge of the molecular weight distribution of a protein in a solution is an extremely important factor in defining a molecular association unit required for expressing the function of the protein. Furthermore, extremely significant problems are encountered when proteins associate or aggregate, often changing their natural state, such as changing their function, or exerting a distinct action different from the expected action (i.e., adverse effect), or loss of stability. For example, monoclonal antibodies intended for use in therapy may exert proper action and stability as long as they are present as a monomer; however, it has been observed that when aggregates form, serious adverse effects may occur, including antigenicity changes and/or deterioration of storage stability (Pharmaceutical Research, 11 (1994), 764-771; Blood, 95 (2000), 1856-1861).
Examination of molecular weight distribution of a protein is regarded as an important technique not only for basic biochemistry research, but also for research and development and quality control of proteinous pharmaceutical products. Known methods for determining molecular weight distribution of a protein include gel filtration chromatography, ultracentrifugal analysis, electrophoretic analysis, light scattering analysis, dynamic light scattering analysis, and the like. Recently, X-ray small angle scattering has also been utilized, and is recognized as a practical technique. However, among these techniques, gel filtration chromatography is most frequently chosen over the other techniques in proteins production laboratories for determining the molecular weight distribution in an aqueous solution. Gel filtration chromatography is considered to be capable of providing information comparable to ultracentrifugal analysis since it employs not only ultraviolet absorption but also light scattering, refractive index, and density in combination (Analytical Biochemistry, 325 (2004), 227-239).
However, gel filtration chromatography continues to present some problems. The elution order of proteins depends on their molecular weight due to diffusion in voids of the stationary phase on the column. The elution order, measured by retention time, is converted into molecular weight. However, the protein not only distributes in the voids of the stationary phase, but also interacts with the stationary phase itself. When the interaction is potent, the elution order may be retarded and may possibly affect the molecular weight information. Because interaction of the aggregates of the protein with the filler is particularly strong, elution from the column in a peak may also be completely inhibited. In such instances, despite of inclusion of the aggregates, an erroneous conclusion that no aggregates are included may be reached by the analysis of the gel filtration chromatography results. It is generally understood that gel filtration chromatography of not just aggregates, but also highly hydrophobic proteins or peptides is difficult. In view of this, it was proposed to optimize the combination of the stationary phase, the mobile phase (developing solvent) and detection method during gel filtration chromatography [Journal of Biochemical and Biophysical Methods, 56 (2003), 1-13]. However, a procedure which alleviates the interaction between protein aggregates, highly hydrophobic proteins, or a highly hydrophobic peptides, and a column stationary phase, so to perfect the recovery in a peak has not been found. As reported by Stulik et al. [Journal of Biochemical and Biophysical Methods, 56 (2003), 1-13], even when an appropriate amount of an additive which is expected to alleviate the interaction between a column stationary phase and a protein, such as for example, an acid, an inorganic salt, or an organic solvent, is added to the mobile phase, improvement of the separation of the aggregates is only occasionally achieved. Furthermore, addition of an appropriate amount of a protein denaturating agent, which is expected to efficaciously alleviate the interaction, into the mobile phase has also been reported; however, difficulty in determining the conditions due to the risk of simultaneous protein degeneration is often encountered. Accordingly, when performing gel filtration chromatography of a protein which may include aggregates, a highly hydrophobic protein, or a highly hydrophobic peptide, it is strongly desirable to be able to easily determine conditions for quantitatively recovering a the aggregates or the hydrophobic protein peak.